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Glycosylation site-specific antibodies and Anti-cancer compounds

a site-specific antibody and anti-cancer technology, applied in the field of characterization of site-specific antibodies for oglcnacylated proteins, can solve the problem that the method cannot identify only the inhibitors of glcnacase, and achieve the effects of increasing the oglcnacylation of c-myc or oglcnacylation of p53, enhancing the oglcnacylation of c-myc and p53

Active Publication Date: 2014-10-02
DETROIT R&D
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides a method for identifying and characterizing the protein O-GlcNAcylation site specificity of an antibody. This allows for the detection and quantitation of the expression of a site-specific O-GlcNAcylated protein in cells and biological samples, as well as the diagnosis of cancer and the screening of anti-cancer compounds. The invention also provides methods for treating cancer by administering an anti-cancer compound that increases O-GlcNAcylation of specific genes. The invention further provides methods for combination therapy of cancer using compounds that target different genes, as well as a method for personalized pancreatic cancer therapy based on sensitivity to one compound.

Problems solved by technology

This method can only identify inhibitors of GlcNAcase that inhibit 4-MU-GlcNAc cleavage.

Method used

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  • Glycosylation site-specific antibodies and Anti-cancer compounds
  • Glycosylation site-specific antibodies and Anti-cancer compounds
  • Glycosylation site-specific antibodies and Anti-cancer compounds

Examples

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example 1

Characterization of Sensitivity of Site-Specific Anti-Peptide Antibodies for O-GlcNAcylated and Corresponding Unmodified p53 and c-Myc Proteins

[0066]Antibody titers were measured with 5,000- through 1,000,000-fold diluted immune sera using ELISA plates coated with free BSA and O-GlcNAcylated and unmodified c-myc or p53 peptides conjugated to BSA (1 μg / well).

[0067]Whereas anti-sera for Ser-149 O-GlcNAcylated (Peptide #1-G) and unmodified (Peptide #1-N) p53 showed almost no cross-reactivity (optical density at 450 nm: 0) with BSA, the immune sera showed very high cross-reactivity (optical density at 450 nm: 3.4 and 3.6, respectively, with 10,000-fold dilution) (FIG. 1, Panel A). Titers (dilution factor at 50% of maximum OD) of the p53 anti-sera were 135,000 and 98,000, respectively.

[0068]Whereas anti-sera for Thr-48 O-GlcNAcylated (Peptide #2-0) and unmodified (Peptide #2-N) c-myc showed almost no cross-reactivity (optical density at 450 nm: 0) with BSA, the immune sera showed very hi...

example 2

Characterization of Specificity of Site-Specific Antibodies for O-GlcNAcylated and Corresponding Unmodified p53 and c-Myc Proteins

[0070]To determine the specificity of the antibodies for O-GlcNAcylated and corresponding unmodified p53 and c-myc proteins, Western blot analysis was carried out with BSA (2 μg / lane) and O-GlcNAcylated and unmodified p53 and c-myc antigen peptides conjugated to BSA (0.2 μg / lane, 10 times less than the BSA, a negative control).

[0071]Specificity of the antibodies was determined by Western blot analysis with BSA conjugated with O-GlcNAcylated and unmodified c-myc and p53 peptides (FIG. 2A). BSA negative controls were produced by simultaneously carrying out conjugations with the EDC or sulfo-SMCC methods but without addition of peptides. Western blot analyses of BSA and BSA conjugated with O-GlcNAcylated and corresponding unmodified c-myc peptides (Peptide #2-0 and #2-N) were carried out using c-myc antibodies. c-Myc antibodies for the O-GlcNAcylated peptide...

example 3

Evidence that the Anti-Peptide Antibodies are O-GlcNAcylation Site-Specific for Ser-149 of p53 and Thr-58 of c-Myc Proteins

[0072]Specificity of the antibodies for O-GlcNAcylated p53 and c-myc proteins was further determined using O-GlcNAcylated p53 and c-myc antigen peptides. Western blot analysis was carried out with BSA (2 μg / lane) and O-GlcNAcylated p53 (Peptide #1-G) and c-myc (Peptide #2-0) antigen peptides conjugated to BSA (0.2 μg / lane, 10 times less than the BSA, a negative control) with antibodies for O-GlcNAcylated p53 and c-myc proteins (FIG. 2B).

[0073]Thr-58 O-GlcNAcylation site-specific c-myc antibodies did not cross-react with O-GlcNAcylated p53 peptides, and Ser-149 O-GlcNAcylation site-specific p53 antibodies did not cross-react with O-GlcNAcylated c-myc peptides (FIG. 2B). This result demonstrated that the O-GlcNAcylation site-specific c-myc and p53 antibodies recognize specifically the Thr-58 and Ser-149 O-GlcNAcylation sites of c-myc and p53, respectively, which a...

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Abstract

A method of characterizing the protein O-GlcNAcylation site-specificity of an antibody. A method of detecting or quantitating the expression of site-specific O-GlcNAcylated proteins expressed in cells and biological samples. A method of diagnosing cancer in a host based on the cellular expression of site-specific O-GlcNAcylated proteins. A method of screening anti-cancer compounds according to their ability to increase a level O-GlcNAcylation of oncogene or tumor suppressor proteins. Methods of treating cancer in an animal host by administering compounds that increase a level of O-GlcNAcylated c-myc or p53 in cancer cells. A method of distinguishing subclasses of pancreatic cancer according to the sensitivity of pancreatic cancer cells to an imidazole derivative, and a method of personalized pancreatic cancer treatment delivered according to the sensitivity subclasses.

Description

GOVERNMENT SUPPORT[0001]Research in this application was supported, in part, by contracts from National Cancer Institute (NCI Contract HHSN261201100073C).BACKGROUND OF THE INVENTION[0002]1. Technical Field[0003]The invention relates to the characterization of site-specific antibodies for O-GlcNAcylated proteins, and for the use of the characterized antibodies for the detection of O-GlcNAcylated proteins in biological systems, the screening of drugs that modulate protein O-GlcNAcylation, and the diagnosis and treatment of cancer.[0004]2. Background Art[0005]O-GlcNAcylation (N-acetylglucosamine modification) of nuclear and cytosol proteins regulates many cell functions. O-GlcNAcylation of proteins plays a critical role in cell cycle regulation, apoptosis and signal transduction. Modification of O-GlcNAcylation of proteins by O-GlcNAc transferase (OTG) (synthesis of O-GlcNAcylated protein) is reversible by the activity of O-GlcNAc hydrolase (O-GlcNAcase) (cleavage of an O-GlcNAc moiety...

Claims

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Application Information

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IPC IPC(8): G01N33/574G01N33/50A61K31/433A61K31/496A61K31/7076
CPCG01N33/57496A61K31/496G01N33/5091A61K31/433A61K31/7076G01N33/5011G01N33/57415G01N33/57419G01N33/57423G01N33/57438G01N2400/02
Inventor KIM, HYESOOKKIM, SOHEE
Owner DETROIT R&D
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